The tissue factor is a start factor related to an extrinsic clotting factor of a blood clotting factor. This tissue factor is one of major components in a reagent for measuring clotting time such as in measurement of prothrombin time. Accordingly, the tissue factor is widely used in clinical diagnosis of abnormality of blood clotting ability.
The tissue factor used as a material of a reagent for measuring clotting time is generally a tissue factor extracted from the brain of cattle, a rabbit or the like. However, a natural tissue factor extracted from such animal brain contains impurities such as blood components, lipoproteins and plasma proteins. Accordingly, when stored for a long time, there is a problem of poor stability to generate precipitates of insoluble materials.
When the naturally derived tissue factor is used as a material of the reagent for measuring clotting time, for improving the stability, proteins such as bovine serum albumin (BSA) and Crystalline (Amano Pharmaceutical Co., Ltd.) and stabilizers such as a surfactant and a highly concentrated glycerin solution are added to the reagent, or the naturally derived tissue factor is partially purified by antibody column chromatography or gel filtration chromatography. However, when a stabilizer such as BSA is added to the reagent, the stabilizer may affect on measuring clotting time. In partial purification, there is a problem that time and costs are required for preparing a large amount of the tissue factor.
With the recent advance of genetic recombination techniques with Escherichia coli, yeast etc. as the host, a genetic recombinant human tissue factor and a genetic recombinant rabbit tissue factor have become commercially available.
However, even if the genetic recombinant tissue factor is used as a material of the reagent, its solution may be liable to denature the protein and to reduce the biological activity depending on its state during storage, and as a consequence, there is a problem of poor storage stability.
A nickel compound-containing reagent for measuring clotting time (JP-A 2001-255332) has already been reported. However, this technique is related to a technique of improving the sensitivity and accuracy of clotting time measurement. Accordingly, there is no description of the stabilization of the reagent for measuring clotting time, particularly the stabilization of the tissue factor.
Generally, a heavy metal such as nickel is known to destabilize a protein. Accordingly, the addition of a chelating agent such as EDTA to a solution containing a protein such as an enzyme is known to remove the influence of a heavy metal (Masato Okada et al., Protein Experimental Note (Upper Volume) Extraction and Separation/Purification, Yodosha, p 24, Sep. 10, 1996). The peroxidation, oxidation and denaturation of a protein by the catalytic action of a heavy metal ion can be prevented by the chelating agent. In addition, hydrolysis with a metalloprotease present in a very small amount in a solution is inhibited by the chelating agent.